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Extremophile organisms are known that can metabolize at temperatures down to - 25 °C (psychrophiles) and up to 122 °C (hyperthermophiles). Understanding viability under extreme conditions is relevant for human health, biotechnological applications, and our search for life elsewhere in the universe. Information about the stability and dynamics of proteins under environmental extremes is an important factor in this regard. Here we compare the dynamics of small Fe-S proteins - rubredoxins - from psychrophilic and hyperthermophilic microorganisms, using three different nuclear techniques as well as molecular dynamics calculations to quantify motion at the Fe site. The theory of 'corresponding states' posits that homologous proteins from different extremophiles have comparable flexibilities at the optimum growth temperatures of their respective organisms. Although 'corresponding states' would predict greater flexibility for rubredoxins that operate at low temperatures, we find that from 4 to 300 K, the dynamics of the Fe sites in these homologous proteins are essentially equivalent., (© 2024. The Author(s).)

The myristoylated calcium sensor SOS3 and its interacting protein kinase, SOS2, play critical regulatory roles in salt tolerance. Mutations in either of these proteins render Arabidopsis thaliana plants hypersensitive to salt stress. We report here the isolation and characterization of a mutant called enh1-1 that enhances the salt sensitivity of sos3-1 and also causes increased salt sensitivity by itself. ENH1 encodes a chloroplast-localized protein with a PDZ domain at the N-terminal region and a rubredoxin domain in the C-terminal part. Rubredoxins are known to be involved in the reduction of superoxide in some anaerobic bacteria. The enh1-1 mutation causes enhanced accumulation of reactive oxygen species (ROS), particularly under salt stress. ROS also accumulate to higher levels in sos2-1 but not in sos3-1 mutants. The enh1-1 mutation does not enhance sos2-1 phenotypes. Also, enh1-1 and sos2-1 mutants, but not sos3-1 mutants, show increased sensitivity to oxidative stress. These results indicate that ENH1 functions in the detoxification of reactive oxygen species resulting from salt stress by participating in a new salt tolerance pathway that may involve SOS2 but not SOS3.

We have used impulsive coherent vibrational spectroscopy (ICVS) to study the Fe(S-Cys)(4) site in oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). In this experiment, a 15 fs visible laser pulse is used to coherently pump the sample to an excited electronic state, and a second <10 fs pulse is used to probe the change in transmission as a function of the time delay. PfRd was observed to relax to the ground state by a single exponential decay with time constants of approximately 255-275 fs. Superimposed on this relaxation are oscillations caused by coherent excitation of vibrational modes in both excited and ground electronic states. Fourier transformation reveals the frequencies of these modes. The strongest ICV mode with 570 nm excitation is the symmetric Fe-S stretching mode near 310 cm(-1), compared to 313 cm(-1) in the low temperature resonance Raman. If the rubredoxin is pumped at 520 nm, a set of strong bands occurs between 20 and 110 cm(-1). Finally, there is a mode at approximately 500 cm(-1) which is similar to features near 508 cm(-1) in blue Cu proteins that have been attributed to excited state vibrations. Normal mode analysis using 488 protein atoms and 558 waters gave calculated spectra that are in good agreement with previous nuclear resonance vibrational spectra (NRVS) results. The lowest frequency normal modes are identified as collective motions of the entire protein or large segments of polypeptide. Motion in these modes may affect the polar environment of the redox site and thus tune the electron transfer functions in rubredoxins.

PF0610, a protein from the hyperthermophile Pyrococcus furiosus, has homologues only in other archaeal species and in three species of Fe(III)-reducing bacteria. It is thought to have a helix-turn-helix (HTH) domain at the N-terminus and possesses two CXXC motifs characteristic of metal binding proteins. We have determined the solution structure of the Zn-bound protein using NMR. PF0610 is a novel winged helix-turn-helix (wHTH) protein with a rubredoxin-like Zn ribbon as its W1 segment. In addition, it possesses a large number of basic residues on its surface. Clusters of basic residues can be found on both helix H3 and the metal-binding loops of W1, suggesting that it might be a DNA-binding protein. Accordingly, gel shift assays using both linear and circular DNA showed that PF0610 does bind DNA, at least in a sequence-independent fashion. Modeling the PF0610-DNA interaction based on other wHTH protein-DNA structures revealed that besides helix H3, basic residues around the second CXXC motif in the metal-binding loop could make extensive contacts with DNA. However, the bulkiness of the W1 region implies that the DNA conformation may be distorted upon PF0610 binding. PF0610 is the first protein known to have a Zn ribbon-embedded wHTH fold and, as such, has potential roles both as a metal-dependent transcription regulator and as a component of the chromosome packing system in P. furiosus. The discovery of this novel structure represents the addition of another branch to the winged HTH protein family and could contribute to our understanding of transcription regulatory processes in P. furiosus.

Rubredoxin from the hyperthermophile Pyrococcus furiosus (Pf Rd) is an extremely thermostable protein, which makes it an attractive subject of protein folding and stability studies. A fundamental question arises as to what the reason for such extreme stability is and how it can be elucidated from a complex set of interatomic interactions. We addressed this issue first theoretically through a computational analysis of the hydrophobic core of the protein and its mutants, including the interactions taking place inside the core. Here we show that a single mutation of one of phenylalanine's residues inside the protein's hydrophobic core results in a dramatic decrease in its thermal stability. The calculated unfolding Gibbs energy as well as the stabilization energy differences between a few core residues follows the same trend as the melting temperature of protein variants determined experimentally by microcalorimetry measurements. NMR spectroscopy experiments have shown that the only part of the protein affected by mutation is the reasonably rearranged hydrophobic core. It is hence concluded that stabilization energies, which are dominated by London dispersion, represent the main source of stability of this protein.

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(S(cys))(4) site in reduced and oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). The oxidized form has also been investigated by resonance Raman spectroscopy. In the oxidized Rd NRVS, strong asymmetric Fe-S stretching modes are observed between 355 and 375 cm(-1); upon reduction these modes shift to 300-320 cm(-1). This is the first observation of Fe-S stretching modes in a reduced Rd. The peak in S-Fe-S bend mode intensity is at approximately 150 cm(-1) for the oxidized protein and only slightly lower in the reduced case. A third band occurs near 70 cm(-1) for both samples; this is assigned primarily as a collective motion of entire cysteine residues with respect to the central Fe. The (57)Fe partial vibrational density of states (PVDOS) were interpreted by normal mode analysis with optimization of Urey-Bradley force fields. The three main bands were qualitatively reproduced using a D(2)(d) Fe(SC)(4) model. A C(1) Fe(SCC)(4) model based on crystallographic coordinates was then used to simulate the splitting of the asymmetric stretching band into at least 3 components. Finally, a model employing complete cysteines and 2 additional neighboring atoms was used to reproduce the detailed structure of the PVDOS in the Fe-S stretch region. These results confirm the delocalization of the dynamic properties of the redox-active Fe site. Depending on the molecular model employed, the force constant K(Fe-S) for Fe-S stretching modes ranged from 1.24 to 1.32 mdyn/A. K(Fe-S) is clearly diminished in reduced Rd; values from approximately 0.89 to 1.00 mdyn/A were derived from different models. In contrast, in the final models the force constants for S-Fe-S bending motion, H(S-Fe-S), were 0.18 mdyn/A for oxidized Rd and 0.15 mdyn/A for reduced Rd. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.

Rubrerythrin was purified by multistep chromatography under anaerobic, reducing conditions from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimer with a molecular mass of 39.2 kDa and contains 2.9 +/- 0.2 iron atoms per subunit. The purified protein had peroxidase activity at 85 degrees C using hydrogen peroxide with reduced P. furiosus rubredoxin as the electron donor. The specific activity was 36 micromol of rubredoxin oxidized/min/mg with apparent K(m) values of 35 and 70 microM for hydrogen peroxide and rubredoxin, respectively. When rubrerythrin was combined with rubredoxin and P. furiosus NADH:rubredoxin oxidoreductase, the complete system used NADH as the electron donor to reduce hydrogen peroxide with a specific activity of 7.0 micromol of H(2)O(2) reduced/min/mg of rubrerythrin at 85 degrees C. Strangely, as-purified (reduced) rubrerythrin precipitated when oxidized by either hydrogen peroxide, air, or ferricyanide. The gene (PF1283) encoding rubrerythrin was expressed in Escherichia coli grown in medium with various metal contents. The purified recombinant proteins each contained approximately three metal atoms/subunit, ranging from 0.4 Fe plus 2.2 Zn to 1.9 Fe plus 1.2 Zn, where the metal content of the protein depended on the metal content of the E. coli growth medium. The peroxidase activities of the recombinant forms were proportional to the iron content. P. furiosus rubrerythrin is the first to be characterized from a hyperthermophile or from an archaeon, and the results are the first demonstration that this protein functions in an NADH-dependent, hydrogen peroxide:rubredoxin oxidoreductase system. Rubrerythrin is proposed to play a role in the recently defined anaerobic detoxification pathway for reactive oxygen species.

The structure of a partially deuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, an organism that grows optimally at 100 degrees C, was determined by using the neutron single-crystal diffractometer dedicated for biological macromolecules (BIX-3) at the JRR-3M reactor of the Japan Atomic Energy Research Institute. Data were collected at room temperature up to a resolution of 1.5 A, and the completeness factor of the data set was 81.9%. The model contains 306 H and 50 D atoms. A total of 37 hydration water molecules were identified, with 15 having all three atoms fully located and the remaining D2O molecules partially defined. The model has been refined to final agreement factors of R = 18.6% and Rfree = 21.7%. Several orientations of the O-D bonds of side chains, whose assignments from x-ray data were previously ambiguous, were clearly visible in the neutron structure. Although most backbone N-H bonds had undergone some degree of H/D exchange throughout the rubredoxin molecule, 5 H atom positions still had distinctly negative (H) peaks. The neutron Fourier maps clearly showed the details of an extensive set of H bonds involving the ND3+ terminus that may contribute to the unusual thermostability of this molecule.

A neutron diffraction study has been carried out at 1.6 A resolution on a mutant rubredoxin from Pyrococcus furiosus using the BIX-3 single-crystal diffractometer at the JRR-3 reactor of the Japan Atomic Energy Research Institute. In order to study the unusual thermostability of rubredoxin from P. furiosus (an organism that grows optimally at 373 K), the hydrogen-bonding patterns were compared between the wild-type protein and a 'triple-mutant' variant. In this mutant protein, three residues were changed (Trp3-->Tyr3, Ile23-->Val23, Leu32-->Ile32) so that they are identical to those in a mesophilic rubredoxin from Clostridium pasteurianum. In the present study, some minor changes were found between the wild-type and mutant proteins in the hydrogen-bonding patterns of the Trp3/Tyr3 region. In this investigation, the H/D-exchange ratios in the protein were also studied. Because the target protein was soaked in D2O during the crystallization procedure, most of the N-H and O-H bonds have become deuterated, while essentially all of the C-H bonds have not. In particular, the H/D-exchange pattern of the N-H amide bonds of the protein backbone is of interest because it may contain some indirect information about the mechanism of unfolding of this small protein. The results are in broad agreement with those from solution NMR studies, which suggest that the backbone amide bonds near the four Cys residues of the FeS4 redox center are most resistant to H/D exchange. Finally, the detailed geometries of the water molecules of hydration around the rubredoxin molecule are also reported. The 1.6 A resolution of the present neutron structure determination has revealed a more detailed picture than previously available of some portions of the water structure, including ordered and disordered O-D bonds. Crystallographic details: space group P2(1)2(1)2(1) (orthorhombic), unit-cell parameters a = 34.48, b = 35.70, c = 43.16 A; final agreement factors R = 0.196 and Rfree = 0.230 for 19,384 observed and 6548 unique neutron reflections collected at room temperature; crystal size 4 mm3; a total of 423 non-H atoms, 290 H atoms and 88 D atoms were located in this study.

Rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus maintains its native structure at high temperatures (373 K). In order to investigate the role of hydrogen bonding, hydration and chain dynamics in this thermostability, wavelength-resolved Laue neutron diffraction data have been collected from the W3Y single mutant (Trp3-->Tyr3) on the spallation neutron protein crystallography station (PCS) at Los Alamos Neutron Science Center. Data were measured at room temperature from nine crystal settings, each of approximately 12 h duration. The total data-measurement period was less than 5 d from a single crystal that had undergone H(2)O/D(2)O exchange. The nominal resolution of the data is 2.1 A.

Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.